PROJECT SUMMARY/ABSTRACT The objectives of this proposal are to understand a new role for cilia in regulating tissue injury responses and to define a mechanism by which cilia dysfunction results in renal cysts. Mice with conditional mutations in genes disrupting cilia formation (e.g., intraflagellar transport 88 (Ift88)) or cilia function (i.e., polycystin 2 (Pkd2)) reveal that the rate of cyst formation depends on the age at which mutations are induced. If disruption occurs in juvenile mice, cysts form uniformly across the kidney within a few weeks. Conversely, if loss is induced in adult mice, cyst formation is slow requiring over six month, with cysts found in localized regions. However, the rate of cyst formation and number of nephrons affected in the adult-induced mutants is increased by renal injury. These data led us to propose that cilia are needed for responses to tissue injury and that the focal cysts in the adult-induced cilia mutants occur in a few nephrons that have received an injury and are unable to undergo normal repair. The cystic kidney phenotype in Pkd2 mutants is significantly more severe than when cilia are disrupted. Thus, it was surprising that Pkd2 mutants that also lack cilia (e.g., Pkd2;Ift88 double mutants) have reduced cystic pathology that is similar to what is observed in the Ift88 mutant alone. This epistatic relationship can be explained by a model in which cilia are needed for both activation and repression of pathway(s) involved in injury and repair. This is identical to the role that cilia play in hedgehog (Hh) signaling. Cilia are needed for Hh, patched, and smoothened to activate the pathway and to generate the Gli activator, but cilia are also needed to generate the Gli3 repressor to turn the pathway off. By analogy, we are proposing a similar relationship between the polycystins and cilia during cyst formation. However, this model does not explain why cysts develop rapidly in non-injured, juvenile-induced mutants. Our studies reveal that macrophage subtypes change during postnatal renal maturation and that the subtypes characteristic of the juvenile period rapidly return following injury. However, in the absence of cilia, this transition process is dysregulated and the response to injury becomes exacerbated or prolonged. Thus, cilia are important for intercellular communication between the epithelium and immune cells and loss of cilia deregulates this injury response driving cyst formation. Through this proposal we will define a novel requirement for cilia following injury, identify and test pathways involved in cilia-regulated injury response, and determine how cilia dysfunction alters intercellular signaling pathways during cyst initiation and expansion. The role of the cilium in the kidney, and many other tissues affected in ciliopathies, has been painfully elusive. Thus, accomplishing the goals of this proposal would be a major advance in understanding of cilia function and the pathogenesis of ciliopathy disorders. Ultimately, this work will identify new therapeutic targets to slow cyst progression in human ciliopathy patients.